The present invention is directed towards specific double knockout(DKO) animals and their use as animal models. More specifically, the double knockout animals contain a disruption in the genes encoding GPX-1 and GPX-GI. Corresponding cells which are amenable to tissue culture are also part of the invention, as are methods of using such cells, including their use as a tool for identifying therapeutic agents. In addition, the invention is directed towards a mouse model of cancer of the small bowel.
Selenium-dependent glutathione peroxidases (GPXs) are a family of enzymes that are most efficient in the reduction of hydroperoxides. At the present time there are four known selenoproteins with GPX activity. These include the classical ubiquitous GPX-1, GPX-GI, the secreted GPX-P, and PHGPX (Ursini, F., Heim, S., Kiess, M., Maiorino, M., Roveri, A., Wissing, J. and Flohe, L. (1999) Science 285, 1393-6). These GPX isozymes are encoded by four distinct genes, the Gpx1 gene for GPX-1, the Gpx2 gene for GPX-GI, the Gpx3 gene for GPX-P, and the Gpx4 gene for PHGPX (Chu, F. F. (1994) Cytogenet Cell Genet 66, 96-8). The GPX-1 and GPX-GI isozymes have very similar properties including substrate specificity and cytosolic localization (Chu, F. F., Doroshow, J. H. and Esworthy, R. S. (1993) J. Biol Chem 268, 2571-6; Esworthy, R. S., Swiderek, K. M., Ho, Y. S. and Chu, F. F. (1998) Biochim Biophys Acta 1381,213-26). The unique feature of GPX-GI is its high levels of expression in the epithelium of the GI-tract. GPX-P is found in body fluids such as plasma, lung and GI-tract (Tham, D. M., Whitin, J. C., Kim, K. K., Zhu, S. X. and Cohen, H. J. (1998) Am J Physiol 275, G1463-71; Kim, K. K., Whitin, J. C., Sukhova, N. M. and Cohen, H. J. (1999) Pediatr Res 46, 715-21). PHGPX is present at a high level in testis and is implicated in sperm maturation (1, Roveri, A., Casasco, A., Maiorino, M., Dalan, P., Cafligaro, A. and Ursini, F. (1992) J Biol Chem 267, 6142-6). PHGPX is present in low level in the GI-tract (Chu, F. F. and Esworthy, R. S. (1995) Arch. Biochem. Biophys. 323, 288-94). Both GPX-P and PHGPX can reduce phospholipid and cholesterol hydroperoxides, while the latter is a more efficient enzyme at reducing these substrates (Thomas, J. P., Maiorino, M., Ursini, F. and Girotti, A. W. (1990) J. Biol. Chem. 265, 454-6 1; Esworthy, R. S., Chu, F. F., Geiger, P., Girotti, A. W. and Doroshow, J. H. (1993) Arch. Biochem. Biophys. 307, 29-34).
It is known that knockout (KO) mice homozygous for disruption of single Gpx1 and Gpx2 genes (i.e., Gpx1-KO and Gpx2-KO mice) display little pathology without additional stress (Ho, Y. S., Magnenat, J. L., Bronson, R. T., Cao, J., Gargano, M., Sugawara, M. and Funk, C. D. (1997) J. Biol. Chem. 272, 16644-51; Esposito, L. A., Kokoszka, J. E., Waymire, K. G., Cottrell, B., MacGregor, G. R. and Wallace, D.C. (2000) Free Radic. Biol. Med. 28, 754-66; Esworthy, R. S., Mann, J. R., Sam, M. and Chu, F. (2000) Am. J. Physiol. Gastrointest. Liver Physiol. 279, G426-G436.). However, aged Gpx1-KO mice display spontaneous weight loss (Esposito, L. A., Kokoszka, J. E., Waymire, K. G., Cottrell, B., MacGregor, G. R. and Wallace, D.C. (2000) Free Radic. Biol. Med. 28, 754-66). For example, the Gpx1 gene is expressed ubiquitously and is highly expressed in the erythrocyte, liver, and kidney. The antioxidant function of GPX-1 is revealed in Gpx1-KO mice especially after treatment with prooxidant chemicals. An increased level of H2O2 is produced from liver mitochondria in older (5-6 month) Gpx1-KO mice compared with that from wildtype mice without any treatment. GPX-1 can prevent lipid peroxidation induced by paraquat herbicide or measured in liver and lung and protect cortical neurons against H2O2 (Cheng, W. H., Ho, Y. S., Valentine, B. A., Ross, D. A., Combs, G. F., Jr. and Lei, X. G. (1998) J. Nutr. 128,1070-6; de Haan, J. B., Bladier, C., Griffiths, P., Kelner, M., O""Shea, R. D., Cheung, N. S., Bronson, R. T., Silvestro, M. J., Wild, S., Zheng, S. S., Beart, P. M., Hertzog, P. J. and Kola, 1. (1998) J. Biol. Chem. 273, 22528-36). The Gpx1-KO mice are more susceptible to neurotoxic agents as malonate, 3-nitropropionic acid, and 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) in the brain (Klivenyi, P., Andreassen, O. A., Ferrante, R. J., Dedeoglu, A., Mueller, G., Lancelot, E., Bogdanov, M., Andersen, J. K., Jiang, D. and Beal, M. F. (2000) J. Neurosci. 20, 1-7). Malonate induces hydroxyl radical generation while MPP+, the active metabolite of MPTP, inhibits mitochondrial complex I activity (Tipton, K. F. and Singer, T. P. (1993) J Neurochem 61, 1191-206). Since these neurotoxins produce oxidative stress and impair energy production, GPX-1 is implicated in protection against oxidative damage.
Depending on the type of insult, lack of GPX activity can also be beneficial under certain circumstances. Mice overexpressing GPX-1 and GPX-P have low levels of peroxides and prostaglandins and are more sensitive to hyperthermia (Mirochnitchenko, O., Palnitkar, U., Philbert, M. and Inouye, M. (1995) Proc. Natl. Acad. Sci. USA 92, 8120-4). We have found that the jejunum crypt of Gpx1-KO mice regenerates better than that in the wildtype after exposure to high dose xcex3-irradiation (Esworthy, R. S., Mann, J. R., Sam, M. and Chu, F. (2000) Am J Physiol Gastrointest Liver Physiol 279, G426). Perhaps the higher level of GPX-GI in the Gpx1-KO mouse intestine are directly responsible for crypt regeneration. Furthermore, the lack of GPX activity in the Gpx1-KO mouse can be protective against kainic acid-induced limbic seizures and neurodegeneration (Jiang, D., Akopian, G., Ho, Y. S., Walsh, J. P. and Andersen, J. K. (2000) Exp. Neurol. 164, 257-68). This appears to result from decreased receptor function for N-methyl-D-aspartate (NMDA), since kainic acid induces NMDA-dependent seizure. Increased GPX activity in mice overexpressing the Gpx1 gene may have enhanced carcinogenic response in skin treated with 7,12-dimethylbenz[a]anthracene and 2-0-tetradecanoylphorbol-13-acetate (Lu, Y. P., Lou, Y. R., Yen, P., Newmark, H. L., Nfirochnitchenko, 0. I., Inouye, M. and Huang, M. T. (1997) Cancer Res. 57, 1468-74). The mechanism of the pro-carcinogenic activity is not known but it is apparent that elevated antioxidant activity can be debilitating, depending on the type of insult.
GPX activity is also implicated in protection against infectious agents. For example, Jaeschke et al. have found that Gpx1-KO mice are more susceptible to neutrophil-mediated parenchymal cell injury during endotoxemia (Jaeschke, H., Ho, Y. S., Fisher, M. A., Lawson, J. A. and Farhood, A. (1999) Hepatology 29,443-50). The galactosamine/endotoxin induced acute liver failure involves neutrophils and GPX protects hepatocytes against peroxides generated by infiltrated neutrophils in the liver. It has also previously been shown that Gpx1-KO mice are more susceptible to coxsackievirus-induced myocarditis (Beck, M. A., Esworthy, R. S., Ho, Y. S. and Chu, F. F. (1998) Faseb J. 12,1143-9). Viral antibody titers in the Gpx1-KO mice are less than 20% of those found in the wildtype mice, suggesting that cellular immune response is impaired in the Gpx1-KO mice.
Gpx2-KO mice have also recently been generated and these mice appear to be normal (Esworthy et al., Am. J. Physiol. Gastrointest. Liver Physiol. 279, G426-G436). Unlike the Gpx1 gene, which is expressed ubiquitously, the Gpx2 gene is expressed specifically in epithelium. The Gpx2 gene is highly expressed in the gastrointestinal tract, and is also present in the breast, lung, and human liver (Chu, F. F., Doroshow, J. H. and Esworthy, R. S. (1993) J. Biol. Chem. 268, 2571-6; Chu, F. F., Esworthy, R. S., Lee, L. and Wilczynski, S. (1999) J. Nutr. 129, 1846-1854). In the GI-epithelium, GPX-1 and GPX-GI contribute to most of GPX activity (Esworthy, R. S., Swiderek, K. M., Ho, Y. S. and Chu, F. F. (1998) Biochim. Biophys. Acta. 1381,213-26.). The lack of pathology in Gpx2-KO mice is not unexpected, since the Gpx2 gene has limited tissue expression, the Gpx1 gene is co-expressed in tissues expressing the Gpx2 gene, and GPX-1 and GPX-GI have similar biochemical and cellular properties.
Reactive oxidative species are implicated to play an important role in the pathogenesis of inflammatory bowel disease (IBD), which is caused, at least in part, by bacterial infection (Panwala, C. M., Jones, J. C. and Viney, J. L. (1998) J Immunol 161, 5733-44; Madsen, K. L., Doyle, J. S., Tavemini, M. M., Jewell, L. D., Rennie, R. P. and Fedorak, R. N. (2000) Gastroenterology 118, 1094-105; Sands, B. E. (2000) Gastroenterology 118, S68-82). IBD consists of two disorders that have similar symptoms, i.e., ulcerative colitis and Crohn""s disease. Although elevated H2O2 is detected in IBD (Simmonds, N. J., Allen, R. E., Stevens, T. R., Van Someren, R. N., Blake, D. R. and Rampton, U. S. (1992) Gastroenterology 103, 186-96; Keshavarzian, A., Sedghi, S., Kanofsky, J., List, T., Robinson, C., Ibrahim, C. and Winship, D. (1992) Gastroenterology 103,177-85)., the protective effect of GPX against IBD has not yet been established. Elevated GPX activity in red blood cells and/or plasma found in IBD patients is implicated against the protective role of GPX in IBD (Thomas, A. G., Miller, V., Shenkin, A., Fell, G. S. and Taylor, F. (1994) J. Pediatr. Gastroenterot. Nutr. 19, 213-9; Offenberg, E. I., Deutsch, J., Smith, S. and Sokol, R. J. (1997) Am J Clin Nutr 65, 1482-8. 31. Rannem, T., Ladefoged, K., Hylander, E., Hegnhoj, J. and Jarnum, S. (1992) Am. J. Clin. Nutr. 56, 933-7). Although selenium-deficiency is commonly present in those patients with severe gastrointestinal disorders (31; Rannem, T., Ladefoged, K., Hylander, E., Hegnhoj, J. and Staun, M. (1998) Scand J Gastroenterol 33, 1057-61), this is believed to result from IBD rather than contributing to IBD.
The evaluation of chemical compounds for potential efficacy as human therapeutics necessitates data and information of a compound""s efficacy in vivo. Ideally, the in vivo system would be human but ethical and pragmatic reasons prevent such data from being accumulated. As an alternative, many laboratory animals provide satisfactory systems for screening potential therapeutics for treating human physiological disorders. Recent advances in recombinant DNA technology have enabled researchers to genetically manipulate the genomes of animals to enhance such animal model systems. For example, the technique of transgenic generation have been utilized to produce knockout mice that do not express a particular endogenous gene.
There presently exists a need for animal models which can be utilized to study the physiological function of GPX activity in the GI-tract. One approach to generate a useful model for such studies would be double knockout (double-KO) mice with a combined disruption of both alleles of each of the Gpx1 and Gpx2 genes.
In accordance with the present invention, an animal model is provided for studying the significance of GPX-1 and GPX-GI, in particular with regard to how these two gene products interact in animal physiology.
In one aspect, the invention provides a transgenic animal deficient in both GPX-1 and GPX-GI activity. In a preferred embodiment, the animal is a mouse. The deficiency is a result of a homozygous double knockout of the Gpx1 and Gpx2 genes in said transgenic mouse.
In another aspect, the invention provides an animal model for the study of pathophysiological function of GPX activity in the ileum and colon in mammals. We have found that the homozygous double-KO mice of the present invention can exhibit symptoms associated with ileitis, colitis, growth retardation, hypothermia, wasting syndrome, inflammatory bowel disease, cancer in the lower GI-tract and leukemia.
In one aspect, the invention provides an animal model for the study of the degree of functional redundancy of GPX-1 and GPX-GI in the ileum and colon in mammals.
In one embodiment, the invention provides transgenic mice which have a homozygous knockout of the Gpx1 gene in said transgenic mice, together with a heterozygous knockout of one allele of the Gpx2 gene. The invention thus provides an animal model for the study of the degree of functional redundancy of GPX-1 and GPX-GI in the ileum and colon.
In another embodiment, the invention provides transgenic mice which have a homozygous knockout of the Gpx2 gene, together with a heterozygous knockout of one allele of the Gpx1 gene in said transgenic mice. The invention thus provides another animal model for the study of the degree of functional redundancy of GPX-1 and GPX-GI in the ileum and colon.
In another embodiment, the invention provides a transgenic double knockout mouse whose genome comprises a homozygous disruption of the endogenous Gpx1 gene and a homozygous disruption of the endogenous Gpx2 gene (a transgenic double knockout Gpx1/Gpx2 mouse), wherein each disruption comprises the insertion of a transgene, and wherein the combined disruptions result in a decreased level of GPX-1 and GPX-GI production and decreased number of cells producing GPX-I and GPX-GI in the transgenic mouse as compared to a nontransgenic mouse.
In another embodiment, the invention provides a transgenic double knockout Gpx1/Gpx2 mouse which exhibits a physiological disease, symptom or symptoms selected from the group consisting of ileitis, colitis, hypothermia, decreased rate of weight gain, perianal ulceration, diarrhea, wasting syndrome, inflammatory bowel disease, cancer of the ileum and myeloleukemia.
In another embodiment, the invention provides a cell or cells isolated from any of the following: a double knockout Gpx1/Gpx2 transgenic mouse, a transgenic mouse having a heterozygous knockout of the Bpx1 gene and a heterozygous knockout of the Gpx2 gene, a transgenic mouse having a homozygous knockout of the Gpx1 gene and a heterozygous knockout of one allele of the Gpx2 gene and a transgenic mouse having a homozygous knockout of the Gpx2 gene and a heterozygous knockout of one allele of the Gpx1.
In one embodiment, the invention provides a transgenic double knockout Gpx1/Gpx2 mouse which further comprises a mouse which is germ free.
In another embodiment, the invention provides the double knockout of the Gpx1 and Gpx2 genes in mice having different genetic backgrounds. The invention thus provides means to identify other genes that affect the severity of ileitis, colitis, inflammatory bowel disease symptoms and progression to cancer.
In another embodiment, the invention provides the double knockout of the Gpx1 and Gpx2 genes in a genetic background of a B6 mouse.
In another aspect, the invention provides a method of selecting an agent for treating a metabolic disorder selected from the group consisting of: ileitis, colitis, hypothermia, decreased rate of weight gain, perianal ulceration, diarrhea, wasting syndrome, inflammatory bowel disease, ileal cancer and myeloleukemia comprising:
(a) measuring a symptom in a knockout mouse whose genome is manipulated to comprise a homozygous disruption of both the endogenous Gpx1 and Gpx2 genes, wherein the disruption of both the Gpx1 and Gpx2 genes results in said knockout mouse exhibiting one of said disease, symptom or symptoms;
(b) administering an agent to said mouse;
(c) measuring one or more of said symptoms in the mouse after administering the agent; and
(d) comparing at least one of said symptoms in the mouse before and after administering the agent, wherein a decrease in said disease, symptom or symptoms after administering the agent indicates the agent is an agent for treating said disease, symptom or symptoms associated with a metabolic disorder.
In another embodiment, the method of observing the effects of treatment of a disease, symptom or symptoms in double-KO Gpx1 and Gpx2 transgenic mice by administering an agent is observed and compared in double-KO Gpx1 and Gpx2 mice having different genetic backgrounds. The method comprises:
(a) measuring a symptom in a first double knockout mouse having a first genetic background, whose genome is manipulated to comprise a homozygous disruption of both the endogenous Gpx1 and Gpx2 genes, wherein the disruption of both the Gpx1 and Gpx2 genes results in said knockout mouse exhibiting a disease, symptom or symptoms selected from the group consisting of: ileitis, colitis, hypothermia, decreased rate of weight gain, perianal ulceration, diarrhea, wasting syndrome, inflammatory bowel disease, ileal cancer and myeloleukemia;
(b) measuring said symptom in a second double knockout mouse having a second genetic background, whose genome is manipulated to comprise a homozygous disruption of both the endogenous Gpx1 and Gpx2 genes, wherein the disruption of both the Gpx1 and Gpx2 genes results in said knockout mouse exhibiting at least one of said disease, symptom or symptoms;
(c) administering an agent to said first and second mouse;
(d) measuring one or more of said symptoms in the first and second mouse after administering the agent; and
(e) comparing at least one of said symptoms in said first and second mouse before and after administering the agent, wherein a decrease in said disease, symptom or symptoms after administering the agent indicates the agent is an agent for treating said disease, symptom or symptoms associated with a metabolic disorder.
In another embodiment, the invention provides a method of observing the effects of treatment of a disease, symptom or symptoms in single and Gpx 1/Gpx 2 double knockout transgenic mice in mice with a B6 genetic background or in hybrid mice having a xc2xd B6, xc2xc 129 SvJ and xc2xc 129S3 genetic background.
In another embodiment, the invention provides a method of selecting an agent that modulates GPX enzyme activity comprising:
(a) administering an agent to a first group of isolated mouse intestinal epithelial cells and not to a second group of mouse intestinal epithelial cells, wherein the genome of both the first and second isolated mouse cell groups has been manipulated to comprise a homozygous disruption of both alleles of the endogenous Gpx1 gene and Gpx2 genes, and wherein the homozygous disruption of both the endogenous Gpx1 gene and Gpx2 genes prevents expression of functional GPX proteins; and
(b) determining the amount of GPX enzyme activity of the first and second cell groups, wherein a difference in the amount of proliferation of the first cell group as compared to the second cell group indicates that the agent modulates GPX enzyme activity. In another embodiment the isolated cells can be observed for a change in level of expression of a marker associated with cancer.
In another embodiment, the invention provides a transgenic animal whose genome contains a homozygous disruption of both the endogenous Gpx1 gene and Gpx2 genes, wherein said animal develops cancer.
In another embodiment, the invention provides an animal model for the development of ileitis, colitis, inflammatory bowel disease, ileal cancer and or myeloleukemia. The model comprises a transgenic animal whose genome comprises a homozygous disruption of the endogenous Gpx1 gene and a homozygous disruption of the endogenous Gpx2 gene, wherein disruption of the Gpx1 and Gpx2 genes is sufficient to effect one or more signs or symptoms in the animal associated with ileitis, colitis, inflammatory bowel disease, ileal cancer and or myeloleukemia.
The invention further provides a method to screen for potential therapeutic agents for the treatment of ileitis, colitis, inflammatory bowel disease, ileal cancer and or myeloleukemia. The method comprises the steps of: a) administering a potential therapeutic agent to a first transgenic animal whose genome comprises a homozygous disruption of both the endogenous Gpx1 gene and Gpx2 genes; b) maintaining the animal for a time sufficient to permit the detection of a change in one or more signs or symptoms in the animal associated with ileitis, colitis, inflammatory bowel disease, ileal cancer and or myeloleukemia in the transgenic animal; c) observing the animal for a change in at least one sign or symptom associated with ileitis, colitis, inflammatory bowel disease, ileal cancer and or myeloleukemia, wherein a second transgenic animal having the same genetic background as the first transgenic animal and whose genome also comprises a homozygous disruption of both the endogenous Gpx1 gene and Gpx2 genes has been maintained under the same conditions as the first animal but has not received the potential therapeutic agent; and d) determining whether one or more signs or symptoms associated with ileitis, colitis, inflammatory bowel disease, ileal cancer and or myeloleukemia is present in the second transgenic animal but not in the first transgenic animal; wherein a potential therapeutic agent will be one that causes a lower incidence of at least one sign or symptom associated with ileitis, colitis, inflammatory bowel disease, ileal cancer and or myeloleukemia in the first transgenic animal.
The invention further provides a method to screen for potential therapeutic agents for the treatment of ileitis, colitis, inflammatory bowel disease, ileal cancer and or myeloleukemia. The method comprises the steps of: a) administering a potential therapeutic agent to an isolated first cell from a first transgenic animal whose genome comprises a homozygous disruption of both the endogenous Gpx1 gene and Gpx2 genes; b) maintaining the cell for a time sufficient to permit the detection of a change in one or more signs or symptoms in the first cell associated with ileitis, colitis, inflammatory bowel disease, ileal cancer and or myeloleukemia in the first cell; c) observing the first cell for a change in at least one sign or symptom associated with ileitis, colitis, inflammatory bowel disease, ileal cancer and or myeloleukemia, wherein a second cell from a second transgenic animal having the same genetic background as the first transgenic animal and whose genome also comprises a homozygous disruption of both the endogenous Gpx1 gene and Gpx2 genes has been maintained under the same conditions as the first cell but has not been exposed to the potential therapeutic agent; and d) determining whether one or more signs or symptoms associated with ileitis, colitis, inflammatory bowel disease, ileal cancer and or myeloleukemia is present in the second transgenic cell but not in the first transgenic cell; wherein a potential therapeutic agent will be one that causes a lower incidence of at least one sign or symptom associated with ileitis, colitis, inflammatory bowel disease, ileal cancer and or myeloleukemia in the first transgenic cell.
The invention further provides a method for assessing the therapeutic effect of a heterologous gene of interest on the development of ileitis, colitis, inflammatory bowel disease, ileal cancer and or myeloleukemia, which comprises the steps of: expressing a heterologous gene of interest in a first transgenic animal whose genome comprises a homozygous disruption of both the endogenous Gpx1 gene and Gpx2 genes; maintaining the first transgenic animal for a time sufficient to permit the detection of one or more signs or symptoms in the first transgenic animal associated with ileitis, colitis, inflammatory bowel disease, ileal cancer and or myeloleukemia in the first transgenic animal; observing the first transgenic animal for a change in at least one sign or symptom associated with ileitis, colitis, inflammatory bowel disease, ileal cancer and or myeloleukemia, wherein a second transgenic animal comprising a homozygous disruption of both the endogenous Gpx1 gene and Gpx2 genes does not express the gene of interest, wherein the second transgenic animal has been maintained under the same conditions as the first transgenic animal; and determining whether one or more signs or symptoms associated with ileitis, colitis, inflammatory bowel disease, ileal cancer and or myeloleukemia is present in the second animal, wherein a gene of interest which reduces the sign or symptom will be one that causes a lower incidence of at least one sign or symptom associated with ileitis, colitis, inflammatory bowel disease, ileal cancer and or myeloleukemia in the first animal.
The invention further provides a method of identifying one or more marker genes or proteins associated with ileal cancer and or myeloleukemia, which comprises the steps of: expressing the marker gene or protein in a first transgenic animal whose genome comprises a homozygous disruption of both the endogenous Gpx1 gene and Gpx2 genes; maintaining the first transgenic animal for a time sufficient to permit the detection a change in one or more signs or symptoms in the first transgenic animal associated with ileitis, colitis, inflammatory bowel disease, ileal cancer and or myeloleukemia in the first transgenic animal; observing the first transgenic animal for a change in at least one sign or symptom associated with ileitis, colitis, inflammatory bowel disease, ileal cancer and or myeloleukemia, wherein a second transgenic animal comprising a homozygous disruption of both the endogenous Gpx1 gene and Gpx2 genes does not express the marker gene or protein, wherein the second transgenic animal has been maintained under the same conditions as the first transgenic animal; and determining whether one or more signs or symptoms associated with ileitis, colitis, inflammatory bowel disease, ileal cancer and or myeloleukemia is present in the first transgenic animal, wherein a marker gene or protein associated with ileitis, colitis, inflammatory bowel disease, ileal cancer and or myeloleukemia will be one that causes a higher incidence of at least one sign or symptom associated with ileitis, colitis, inflammatory bowel disease, ileal cancer and or myeloleukemia in the first animal.
In particularly preferred embodiments, the first and second transgenic animals utilized in the methods of the invention are mice.